Molecular reaction mechanisms of heterotrimeric G-Proteins

异源三聚体g蛋白是细胞信号通路中的中心开关。这些通常是通过g蛋白偶联受体（GPCR）开启的，它催化从GDP到GTP的交换。这导致异三聚体复合物分解成其亚基，并激活重要的信号通路。它们在α亚基内被GTP水解再次关闭。由于其在药理学上的重要性，目前世界各地的许多研究小组都在研究gpcr。相比之下，我们将使用时间分辨FTIR差分光谱和生物分子模拟，通过广泛的综合方法研究较少详细的galpha蛋白水解。alpha-亚基由g结构域和一个额外的全α结构域组成，g结构域在小的gtpase中也保守。从x射线晶体学中获得的Galpha的几种蛋白质结构已经可用。然而，不可水解的GTP类似物被使用，严重干扰催化中心。互补的，时间分辨的ftir光谱可以解析蛋白质在生理条件下的动力学及其与天然核苷酸的原子细节的相互作用。我们已经成功地将这种方法应用于许多小型gtp。在我们的初步工作中，我们能够将这种方法也转移到α蛋白上。为了从红外光谱中解码详细的分子信息，还应用了QM/MM计算。这里我们要阐明催化重要氨基酸的作用。除了谷氨酰胺，稳定亲核水，特别是催化精氨酸的作用将被确定。后者与小gtpase相反，是催化中心内的内在残留物。此外，我们想了解rgs蛋白如何不像小gtpase的gap，能够进一步加速水解，而不直接与核苷酸相互作用。这调节了信号通路的中断。除了野生型蛋白外，我们还想确定由精氨酸和谷氨酰胺突变引起的蛋白质功能障碍。这些突变在麦昆-奥尔布赖特综合征和癌症等严重疾病中起着重要作用。我们将从抑制性Gi和激活性Gs开始我们的研究。随后计划研究Gq和Gt，以阐明机制上的变化和相似之处。其他疾病如霍乱和百日咳是由毒素介导的，通过adp核糖基化修饰α -蛋白。本文将详细探讨这种改性的机理。

Heterotrimeric G-proteins are central switches within signalling pathways in cells. These are usually switched on via G-protein coupled receptors (GPCR), which catalyse the exchange from GDP to GTP. This causes the breakup of the heterotrimeric complex into its subunits and activates important signalling pathways. They are switched off again by GTP hydrolysis within the Galpha-subunit. GPCRs are currently investigated by numerous research groups worldwide, especially due to their importance in pharmacology. In contrast, we will investigate the less detailed studied hydrolysis of Galpha-proteins by a broad, integrative approach using time-resolved FTIR difference-spectroscopy and biomolecular simulations. The Galpha-subunits consist of the G-domain, conserved also in small GTPases, and an additional all-alpha domain. Several protein structures of Galpha obtained from X-ray crystallography are already available. However, non-hydrolysable GTP analogues were used, which interfere seriously with the catalytic centre. Complementary, time-resolved FTIR-spectroscopy can resolve the dynamics of the protein under physiological conditions and its interaction with atomic detail using the natural nucleotide. We have applied this approach already successfully for many small GTPases. In our preliminary work, we were able to transfer this approach also to Galpha-proteins. In order to decode detailed molecular information from the IR spectra, QM/MM calculations are applied in addition. Here we want to elucidate the role of the catalytically important amino acids. Besides a glutamine, stabilizing the nucleophilic water, in particular the role of the catalytic arginine will be determined. The latter is, in contrast to small GTPases, an intrinsic residue within the catalytic centre. Further, we want to understand how RGS-proteins, unlike GAPs of small GTPases, are able to further accelerate hydrolysis, without direct interaction with the nucleotide. This regulates the interruption of signalling pathways. Besides wildtype protein, we want to determine especially dysfunctions of the protein induced by mutations of the arginine and the glutamine. These mutations play an important role in severe diseases as the McCune-Albright syndrome and cancer. We will start our investigations with the inhibitory Gi and the activating Gs. Later it is planned to investigate Gq and Gt, to elucidate mechanistic alterations and similarities. Further diseases like cholera and pertussis are mediated by toxins, modifying Galpha-proteins by ADP-ribosylation. The mechanism of this modification will be investigated in detail.